The orientation of rhodopsin and a 220,000 M.W. glycoprotein ROS 1.2 was studied using 125I-lectins in combination with proteolytic enzymes. Freshly prepared sealed discs bound low quantities of 125I-Concanavalin A (Con A) and 125I-wheat germ agglutinin (WGA). When the discs were unsealed by treatment with low concentration on Triton X-100 or sodium cholate, a 25-30 fold increase in 125I-lectin binding was observed. An increase in lectin binding was also observed when discs were unsealed by freezing and thawing prior to binding studies. SDS-gel electrophoresis in combination with fluorescent lectin binding studies indicated that both rhodopsin and ROS 1.2 bind Con A and WGA. Trypsin and pronase treatment of sealed discs had no effect on the subsequent binding of lectins in the presence or absence of the detergent. SDS-gel electrophoresis, however, showed that both these membrane proteins were degraded to lower molecular weight fragments by these enzymes. From these results, we conclude that the carbohydrate chains of rhodopsin and ROS 1.2 are oriented toward the interior of disc membranes and inaccessible to lectin binding. Regions of the polypeptide chains of these proteins, however, are exposed on the external or cytoplasmic surface of discs and susceptible to degradation by proteases. Thus, rhodopsin and ROS 1.2 appear to be transmembrane glycoproteins.